Nonalcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, is rapidly becoming a major public health crisis in the United States paralleling the epidemic of obesity and diabetes. It has been speculated that the burden of NAFLD among the Veteran population is poised to exceed that of the general US population. The number of Veterans diagnosed with NAFLD increased nearly 3-fold from 2003 to 2011 with the steepest rise among Veterans under 45 years of age. Furthermore, Veterans with NAFLD have a higher prevalence of risk factors associated with progression to cirrhosis. Unfortunately there is no definitive pharmacologic therapy to prevent or treat NAFLD owing to the fact that the fundamental pathogenesis of this disorder is incompletely understood. In the current proposal we identify plasminogen activator inhibitor 1 (PAI-1) as a novel therapeutic target for NAFLD. PAI-1, best characterized as a regulator of the fibrinolytic cascade, is increasingly recognized as a mediator of metabolic diseases such obesity and diabetes. Patients with NAFLD have significantly elevated circulating PAI-1 levels and the degree of elevation closely correlates with the severity of liver disease. Despite this strong association, it remains unknown whether PAI-1 functions in the pathogenesis of NAFLD or whether pharmacologic inhibition of PAI-1 may have therapeutic potential. We have generated compelling preliminary data demonstrating that genetic deletion or pharmacologic inhibition of PAI-1 protects against the development of hepatic steatosis in mice. In the current proposal we assert that PAI-1 has a fundamental mechanistic role in the progression of NAFLD and inhibiting PAI-1 will be an effective therapeutic strategy to reverse NAFLD in mice. In Specific Aim 1 we will firmly establish the role of PAI-1 in NAFLD progression and investigate the therapeutic potential of a novel small molecule PAI-1 inhibitor in a murine dietary model of nonalcoholic steatohepatitis (NASH). To determine the role of PAI-1 in progressive NASH (characterized by hepatic inflammation and fibrosis) mice bearing a global deletion of Pai-1 will be challenged with a high-fat, high-fructose corn syrup (a.k.a ?fast-food diet?). To determine whether inhibiting PAI-1 effectively prevents NASH progression, wild-type C57Bl/6 mice will be fed a fast-food diet supplemented with TM5441, a novel, orally-active, highly-specific small molecular inhibitor of PAI-1. To determine whether pharmacologic inhibition of PAI-1 reverses existing NAFLD, TM5441 will be administered to mice with experimentally induced NAFLD. We hypothesize that 1) Pai-1 deletion or PAI-1 inhibition will protect against fast-food diet-induced steatohepatitis and fibrosis and 2) pharmacologic inhibition of PAI-1 using TM5441 will effectively reduce existing steatosis in mice. In Specific Aim 2 we will determine the function of adipose-derived Pai-1 in the development of murine NAFLD and obesity. Adipose tissue is a major source of circulating PAI-1 yet the function of PAI-1 as an adipokine is poorly understood. Our preliminary studies indicate that global deletion, but not hepatocyte-specific deletion of Pai-1, protects against diet-induced hepatic steatosis. These data raise the hypothesis that adipose-derived Pai-1 may be a primary driver of NAFLD in mice. To determine the function of adipose-derived Pai-1, we will generate mice bearing an adipocyte-specific deletion of Pai-1. We will then examine the effects of adipocyte-specific deletion on diet- induced NAFLD, obesity, and adipose tissue inflammation in mice. Through the proposed studies we will definitively establish a causative role for PAI-1 in the progression of murine NASH, determine the tissue- specific function of Pai-1 in murine metabolic disease, and identify PAI-1 as a novel therapeutic target for NAFLD. We anticipate that the present work will serve as the platform to investigate PAI-1 inhibition as a therapeutic strategy for Veterans with NAFLD.